Lettuce named DESERT EAGLE

ABSTRACT

Novel lettuce, such as lettuce designated DESERT EAGLE is disclosed. In some embodiments, the invention relates to the seeds of lettuce DESERT EAGLE, to the plants and plant parts of lettuce DESERT EAGLE, and to methods for producing a lettuce plant by crossing the lettuce DESERT EAGLE with itself or another lettuce plant. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce plants derived from the lettuce DESERT EAGLE.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to, and the benefits of U.S.Provisional Patent Application No. 62/221,220, filed Sep. 21, 2015,which is herein incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive lettuce (Lactuca sativa) cultivar, such as cultivardesignated DESERT EAGLE, and to methods of making and using such plants.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Lettuce is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding lettucecultivar that are agronomically sound. The reasons for this goal are tomaximize the amount of yield produced on the land used as well as toimprove the plant agronomic qualities. To accomplish this goal, thelettuce breeder must select and develop lettuce plants that have thetraits that result in superior cultivars.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments, there is provided onenovel lettuce cultivar, designated DESERT EAGLE. This invention thusrelates to the seeds of lettuce cultivar designated DESERT EAGLE, to theplants or parts thereof of lettuce cultivar designated DESERT EAGLE, toplants or parts thereof consisting essentially of the phenotypic andmorphological characteristics of lettuce cultivar designated DESERTEAGLE, and/or having all the physiological and morphologicalcharacteristics of lettuce cultivar designated DESERT EAGLE and/orhaving the characteristics of lettuce cultivar designated DESERT EAGLElisted in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental condition,and/or having one or more of the physiological and morphologicalcharacteristics of lettuce cultivar designated DESERT EAGLE listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental condition and/orhaving all the physiological and morphological characteristics oflettuce cultivar designated DESERT EAGLE listed in Table 1 when grown inthe same environmental condition. The invention also relates tovariants, mutants and trivial modifications of the seed or plant oflettuce cultivar designated DESERT EAGLE.

Plant parts of the lettuce cultivar of the present invention are alsoprovided, such as a head, leaf, flower, cell, pollen or ovule obtainedfrom the plant cultivar. The present invention provides heads and/orleaves of the lettuce cultivar of the present invention. Such headsand/or leaves could be used as fresh products for consumption or inprocesses resulting in processed products such as food productscomprising one or more harvested part of the lettuce plant DESERT EAGLE,for example harvested leaves and/or heads. The harvested part or foodproduct can be or can comprise the lettuce head and/or leaves of thelettuce plant DESERT EAGLE or a salad mixture comprising leaves of thelettuce plant DESERT EAGLE. The food products might have undergone oneor more processing steps such as, but not limited to cutting, washing,mixing, etc. All such products are part of the present invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of the United States of America, i.e., avariety that is predominantly derived from lettuce cultivar designatedDESERT EAGLE or from a variety that i) is predominantly derived fromlettuce cultivar designated DESERT EAGLE, while retaining the expressionof the essential characteristics that result from the genotype orcombination of genotypes of lettuce cultivar designated DESERT EAGLE;ii) is clearly distinguishable from lettuce cultivar designated DESERTEAGLE; and iii) except for differences that result from the act ofderivation, conforms to the initial variety in the expression of theessential characteristics that result from the genotype or combinationof genotypes of the initial variety or cultivar.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture oflettuce cultivar designated DESERT EAGLE. In some embodiments, thetissue culture is capable of regenerating plants consisting essentiallyof the phenotypic and morphological characteristics of lettuce cultivardesignated DESERT EAGLE, and/or having all the phenotypic andmorphological characteristics of lettuce cultivar designated DESERTEAGLE, and/or having the physiological and morphological characteristicsof lettuce cultivar designated DESERT EAGLE, and/or having thecharacteristics of lettuce cultivar designated DESERT EAGLE. In someembodiments, the plant parts and cells used to produce such tissuecultures can be embryos, meristematic cells, seeds, callus, pollen,leaves, anthers, pistils, roots, root tips, stems, petioles, heads,cotyledons, hypocotyls, ovaries, seed coat, fruits, endosperm, flowers,axillary buds or the like. Protoplasts produced from such tissue cultureare also included in the present invention. The lettuce shoots, rootsand whole plants regenerated from the tissue culture, as well as theheads and leaves produced by said regenerated plants are also part ofthe invention. In some embodiments, the whole plants regenerated fromthe tissue culture have one, more than one, or all of the physiologicaland morphological characteristics of lettuce cultivar designated DESERTEAGLE listed in Table 1, including but not limited to when grown in thesame environmental condition.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In some embodiments, the methodscomprise collecting a part of a lettuce cultivar designated DESERT EAGLEand regenerating a plant from said part. In some embodiments, the partcan be for example a leaf cutting that is rooted into an appropriatemedium according to techniques known by the one skilled in the art.Plants, plant parts and heads thereof produced by such methods are alsoincluded in the present invention. In another aspect, the plants andheads thereof produced by such methods consist essentially of thephenotypic and morphological characteristics of lettuce cultivardesignated DESERT EAGLE, and/or having all the phenotypic andmorphological characteristics of lettuce cultivar designated DESERTEAGLE, and/or having the physiological and morphological characteristicsof lettuce cultivar designated DESERT EAGLE, and/or having thecharacteristics of lettuce cultivar designated DESERT EAGLE. In someembodiments, plants produced by such methods consist of one, more thanone, or all phenotypic and morphological characteristics of lettucecultivar designated DESERT EAGLE listed in Table 1, including but notlimited to when grown in the same environmental condition.

Further included in the invention are methods for producing heads fromthe lettuce cultivar designated DESERT EAGLE. In some embodiments, themethods comprise growing a lettuce cultivar designated DESERT EAGLE toproduce a lettuce head. In some embodiments, the methods furthercomprise harvesting the lettuce head. Such lettuce heads and leavesthereof are part of the present invention.

Also included in this invention are methods for producing a lettuceplant. In some embodiments, the lettuce plant is produced by crossingthe lettuce cultivar designated DESERT EAGLE with itself or anotherlettuce plant. In some embodiments, the other plant can be a lettucehybrid or line. When crossed with itself, i.e. when DESERT EAGLE iscrossed with another lettuce cultivar DESERT EAGLE or self-pollinated,lettuce cultivar DESERT EAGLE will be conserved (e.g. as an inbred).When crossed with another, different lettuce plant, an F1 hybrid seed isproduced if the different lettuce plant is an inbred and a “three-waycross” seed is produced if the different lettuce plant is a hybrid. SuchF1 hybrid seed and three-way hybrid seeds and plants produced by growingsaid F1 and three-way hybrid seeds are included in the presentinvention. Methods for producing a F1 and three-way hybrid lettuce seedcomprising crossing lettuce cultivar DESERT EAGLE lettuce plant with adifferent lettuce line or hybrid and harvesting the resultant hybridlettuce seed are also part of the invention. The hybrid lettuce seedsproduced by the methods comprising crossing lettuce cultivar DESERTEAGLE lettuce plant with a different lettuce plant and harvesting theresultant hybrid lettuce seed are included in the invention, as areincluded the hybrid lettuce plants or parts thereof and seeds producedby said grown hybrid lettuce plants.

Further included in the invention are methods for producing a lettuceseed and plants made thereof. In some embodiments, said methods compriseself-pollinating the lettuce cultivar DESERT EAGLE and harvesting theresultant seeds. Lettuce seeds produced by such method are also part ofthe invention.

In another embodiment, this invention also relates to methods forproducing other lettuce plants derived from lettuce cultivar DESERTEAGLE and to the lettuce plants derived by the use of those methods.

In some embodiments, such methods for producing a lettuce plant derivedfrom the lettuce cultivar DESERT EAGLE comprise (a) self-pollinating thelettuce cultivar DESERT EAGLE plant at least once to produce a progenyplant derived from lettuce cultivar DESERT EAGLE; In some embodiments,the methods further comprise (b) crossing the progeny plant derived fromlettuce cultivar DESERT EAGLE with itself or a second lettuce plant toproduce a seed of a progeny plant of a subsequent generation; In someembodiments, the methods further comprise (c) growing the progeny plantof the subsequent generation. In some embodiment, the method furthercomprises (d) crossing the progeny plant of the subsequent generationwith itself or a second lettuce plant to produce a lettuce plant derivedfrom the lettuce cultivar DESERT EAGLE. In further embodiments, stepsb), step c) and/or step d) are repeated for at least 1, 2, 3, 4, 5, 6,7, 8, or more generation to produce a lettuce plant derived from thelettuce cultivar DESERT EAGLE. In some embodiments, within each crossingcycle, the second plant is the same plant as the second plant in thelast crossing cycle. In some embodiment, within each crossing cycle, thesecond plant is different from the second plant of the last crossingcycle.

Another method for producing a lettuce plant derived from the varietyDESERT EAGLE, comprises the steps of: (a) crossing the DESERT EAGLEplant with a second lettuce plant to produce a progeny plant derivedfrom lettuce cultivar DESERT EAGLE; In some embodiments, the methodfurther comprises (b) crossing the progeny plant derived from lettucecultivar DESERT EAGLE with itself or a second lettuce plant to produce aseed of a progeny plant of a subsequent generation; In some embodiments,the method further comprises (c) growing the progeny plant of thesubsequent generation from the seed; In some embodiments, the methodfurther comprises (d) crossing the progeny plant of the subsequentgeneration with itself or a second lettuce plant to produce a lettuceplant derived from DESERT EAGLE. In a further embodiment, steps b), step(c) and/or step (d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, ormore generation to produce a lettuce plant derived from DESERT EAGLE. Insome embodiments, within each crossing cycle, the second plant is thesame plant as the second plant in the last crossing cycle. In someembodiments, within each crossing cycle, the second plant is differentfrom the second plant in the last crossing cycle.

In another aspect, the present invention provides methods of introducingor modifying one or more desired trait(s) into the lettuce cultivarDESERT EAGLE and plants or seeds obtained from such methods. The desiredtrait(s) may be, but not exclusively, a single gene. In someembodiments, the gene is a dominant allele. In some embodiments, thegene is a partially dominant allele. In some embodiments, the gene is arecessive allele. In some embodiments, the transferred gene or geneswill confer such traits as male sterility, herbicide resistance, insectresistance, resistance for bacterial, fungal, mycoplasma or viraldisease, improved shelf life, water-stress tolerance, delayed senescenceor controlled ripening, enhanced plant quality such as improved droughtor salt tolerance, enhanced plant vigor, improved or changed colors orimprove fresh cut application. For the present invention and the skilledartisan, disease is understood to include, but not limited to fungaldiseases, viral diseases, bacterial diseases, mycoplasm diseases, orother plant pathogenic diseases and a disease resistant plant willencompass a plant resistant to fungal, viral, bacterial, mycoplasm, andother plant pathogens. The gene or genes may be naturally occurringlettuce gene(s), mutant(s) or) or genes modified through New BreedingTechniques. In some embodiments, the method for introducing the desiredtrait(s) is a backcrossing process making use of a series of backcrossesto lettuce cultivar DESERT EAGLE during which the desired trait(s) ismaintained by selection. The single gene conversion plants that can beobtained by the method are included in the present invention.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as lettuce cultivarDESERT EAGLE. Alternatively, if the trait is not modified into eachnewly developed line/cultivar such as lettuce cultivar DESERT EAGLE,another typical method used by breeders of ordinary skill in the art toincorporate the modified gene is to take a line already carrying thegene and to use such line as a donor line to transfer the gene into thenewly developed line. The same would apply for a naturally occurringtrait or one arising from spontaneous or induced mutations.

In some embodiments, the backcross breeding process of lettuce cultivarDESERT EAGLE comprises (a) crossing lettuce cultivar DESERT EAGLE withplants that comprise the desired trait(s) to produce F1 progeny plants.In some embodiments, the process further comprises (b) selecting the F1progeny plants that have the desired trait(s); In some embodiments, theprocess further comprises (c) crossing the selected F1 progeny plantswith the lettuce cultivar DESERT EAGLE plants to produce backcrossprogeny plants; In some embodiments, the process further comprises (d)selecting for backcross progeny plants that have the desired trait(s)and physiological and morphological characteristics of the lettucecultivar DESERT EAGLE to produce selected backcross progeny plants; Insome embodiments, the process further comprises (e) repeating steps (c)and (d) one, two, three, four, five six, seven, eight, nine or moretimes in succession to produce selected, second, third, fourth, fifth,sixth, seventh, eighth, ninth or higher backcross progeny plants thatconsist essentially of the phenotypic and morphological characteristicsof the lettuce cultivar DESERT EAGLE and/or have all the phenotypic andmorphological characteristics of the lettuce cultivar DESERT EAGLE,and/or have the desired trait(s) and the physiological and morphologicalcharacteristics of the lettuce cultivar DESERT EAGLE as determined inTable 1, including but not limited to at a 5% significance level whengrown in the same environmental conditions. In some embodiments, thebackcross breeding process of lettuce cultivar of DESERT EAGLE comprisesthe following steps: (a) crossing lettuce cultivar DESERT EAGLE withplants of another line that comprises the desired trait(s) to produce F1progeny plants, (b) selecting the F1 progeny plants that have thedesired trait(s); (c) crossing the selected F1 progeny plants with thelettuce cultivar DESERT EAGLE plants to produce backcross progenyplants; (d) selecting for backcross progeny plants that have the desiredtrait(s) and physiological and morphological characteristics of thelettuce cultivar DESERT EAGLE to produce selected backcross progenyplants; and (e) repeating steps (c) and (d) one, two, three, four, fivesix, seven, eight, nine or more times in succession to produce selected,second, third, fourth, fifth, sixth, seventh, eighth, ninth or higherbackcross progeny plants that consist essentially of the phenotypic andmorphological characteristics of the lettuce cultivar DESERT EAGLE,and/or have all the phenotypic and morphological characteristics of thelettuce cultivar DESERT EAGLE, and/or have the desired trait(s) and thephysiological and morphological characteristics of the lettuce cultivarDESERT EAGLE as determined in Table 1, including but not limited to at a5% significance level when grown in the same environmental conditions.The lettuce plants or seed produced by the methods are also part of theinvention. Backcrossing breeding methods, well known to one skilled inthe art of plant breeding will be further developed in subsequent partsof the specification.

In an embodiment of this invention is a method of making a backcrossconversion of lettuce cultivar DESERT EAGLE. In some embodiments, themethod comprises crossing lettuce cultivar DESERT EAGLE with a donorplant comprising a mutant gene(s), a naturally occurring gene(s), or agene(s) and/or sequences modified through the use of New BreedingTechniques conferring one or more desired trait to produce F1 progenyplant. In some embodiment, the method further comprises selecting the F1progeny plant comprising the naturally occurring gene(s), mutant gene(s)or modified gene(s) and/or sequences conferring the one or more desiredtrait. In some embodiments, the method further comprises backcrossingthe selected progeny plant to the lettuce cultivar DESERT EAGLE. Thismethod may further comprise the step of obtaining a molecular markerprofile of the lettuce cultivar DESERT EAGLE and using the molecularmarker profile to select for a progeny plant with the desired trait andthe molecular marker profile of the lettuce cultivar DESERT EAGLE. Theplants or parts thereof produced by such methods are also part of thepresent invention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the lettuce cultivar DESERT EAGLE is at least 1, 2, 3,4, 5 or more. A single locus may contain one or several genes A singlelocus conversion also allows for making one or more site specificchanges to the plant genome, such as, without limitation, one or morenucleotide change, deletion, insertions, etc. In some embodiments, thesingle locus conversion is performed by genome editing, a.k.a. genomeediting with engineered nucleases (GEEN). In some embodiments, thegenome editing comprises using one or more engineered nucleases. In someembodiments, the engineered nucleases include, but are not limited toZinc finger nucleases (ZFNs), Transcription Activator-Like EffectorNucleases (TALEN5), the CRISPR/Cas system, engineered meganucleasere-engineered homing endonucleases, and endonucleases for DNA guidedgenome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547). In some embodiments, the single locus conversionchanges one or several nucleotides of the plant genome. Such genomeediting techniques are some of the techniques now known by a personskilled in the art and herein are collectively referred to as ‘NewBreeding Techniques’.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding but not limited to, recurrent selection, backcrossing,pedigree breeding, molecular marker (Isozyme Electrophoresis,Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), and Simple Sequence Repeats (SSRs) which are also referred toas Microsatellites, Single Nucleotide Polymorphisms (SNPs), etc.)enhanced selection, genetic marker enhanced selection andtransformation. Seeds, lettuce plants, and parts thereof produced bysuch breeding methods are also part of the invention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the lettuce cultivar DESERT EAGLE.Variants, mutants and trivial modifications of the seed or plant oflettuce cultivar DESERT EAGLE can be generated by methods available toone skilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisense,RNA interference and other techniques such as the New BreedingTechniques. For more information of mutagenesis in plants, such asagents, protocols, see Acquaah et al. (Principles of plant genetics andbreeding, Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464, whichis herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the lettucecultivar DESERT EAGLE and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newlettuce plants which comprises one or more or all of the morphologicaland physiological characteristics of lettuce cultivar DESERT EAGLE. Insome embodiments, the new lettuce plants obtained from the screeningprocess comprise all of the morphological and physiologicalcharacteristics of the lettuce cultivar DESERT EAGLE, and one or moreadditional or different morphological and physiological characteristicsthat lettuce cultivar DESERT EAGLE does not have.

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein either the first or second parent lettuce plant is alettuce cultivar DESERT EAGLE. Further, both first and second parentlettuce plants can come from the lettuce cultivar DESERT EAGLE. Further,the lettuce cultivar DESERT EAGLE can be self-pollinated i.e. the pollenof a lettuce cultivar DESERT EAGLE can pollinate the ovule of the samelettuce cultivar DESERT EAGLE, respectively. When crossed with anotherlettuce plant, a hybrid seed is produced. Such methods of hybridizationand self-pollination are well known to those skilled in the art ofbreeding.

A lettuce cultivar such as lettuce cultivar DESERT EAGLE has beenproduced through several cycles of self-pollination and is therefore tobe considered as a homozygous plant or line. An inbred line can also beproduced though the dihaploid system which involves doubling thechromosomes from a haploid plant or embryo thus resulting in an inbredline that is genetically stable (homozygous) and can be reproducedwithout altering the inbred line: Haploid plants could be obtained fromhaploid embryos that might be produced from microspores, pollen, anthercultures or ovary cultures. The haploid embryos may then be doubled bychemical treatments such as by colchicine or be doubled autonomously.The haploid embryos may also be grown into haploid plants and treated toinduce the chromosome doubling. In either case, fertile homozygousplants are obtained. A hybrid variety is classically created through thefertilization of an ovule from an inbred parental line by the pollen ofanother, different inbred parental line. Due to the homozygous state ofthe inbred line, the produced gametes carry a copy of each parentalchromosome. As both the ovule and the pollen bring a copy of thearrangement and organization of the genes present in the parental lines,the genome of each parental line is present in the resulting F1 hybrid,theoretically in the arrangement and organization created by the plantbreeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a man skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga lettuce cultivar DESERT EAGLE-derived lettuce plant by crossinglettuce cultivar DESERT EAGLE with a second lettuce plant. In someembodiments, the method further comprises obtaining a progeny seed fromthe cross. In some embodiment, the method further comprises growing theprogeny seed, and possibly repeating the crossing and growing steps withthe lettuce cultivar DESERT EAGLE-derived plant from 0 to 7, or moretimes. Thus, any such methods using the lettuce cultivar DESERT EAGLEare part of this invention: selfing, backcrosses, hybrid production,crosses to populations, and the like. All plants produced using lettucecultivar DESERT EAGLE as a parent are within the scope of thisinvention, including plants derived from lettuce cultivar DESERT EAGLE.

Such plants might exhibit additional and desired characteristics ortraits such as high seed yield, high seed germination, seedling vigor,early maturity, high yield, disease tolerance or resistance, andadaptability for soil and climate conditions. Consumer-driven traits,such as a preference for a given head size, shape, color, texture,taste, are other traits that may be incorporated into new lettuce plantsdeveloped by this invention.

A lettuce plant can also be propagated vegetatively. A part of theplant, for example a shoot tissue, is collected, and a new plant isobtained from the part. Such part typically comprises an apical meristemof the plant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including for example rooting or developmentof shoots. This is achieved using methods well-known in the art.Accordingly, in one embodiment, a method of vegetatively propagating aplant of the present invention comprises collecting a part of a plantaccording to the present invention, e.g. a shoot tissue, and obtaining aplantlet from said part. In one embodiment, a method of vegetativelypropagating a plant of the present invention comprises: a) collectingtissue of a plant of the present invention; b) rooting said proliferatedshoots to obtain rooted plantlets. In one embodiment, a method ofvegetatively propagating a plant of the present invention comprises: a)collecting tissue of a plant of the present invention; b) cultivatingsaid tissue to obtain proliferated shoots; c) rooting said proliferatedshoots to obtain rooted plantlets. In one embodiment, such methodfurther comprises growing a plant from said plantlets. In oneembodiment, a head is harvested from said plant. In one embodiment, thehead is processed into products prepared cut heads and leaves.

In some embodiments, the present invention teaches a seed of lettucecultivar DESERT EAGLE, wherein a representative sample of seed of saidlettuce cultivar is deposited under NCIMB No. 42806.

In some embodiments, the present invention teaches a lettuce plant, or apart thereof, produced by growing the deposited DESERT EAGLE seed.

In some embodiments, the present invention teaches lettuce plant parts,wherein the lettuce part is selected from the group consisting of: aleaf, a flower, a head, an ovule, pollen, and a cell.

In some embodiments, the present invention teaches a lettuce plant, or apart thereof, having all of the characteristics of lettuce cultivarDESERT EAGLE as listed in Table 1 of this application including but notlimited to when grown in the same environmental conditions.

In some embodiments, the present invention teaches a lettuce plant, or apart thereof, having all of the physiological and morphologicalcharacteristics of lettuce cultivar DESERT EAGLE, wherein arepresentative sample of seed of said lettuce plant was deposited underNCIMB No. 42806.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited lettuce cultivar DESERT EAGLE seed, wherein cells of thetissue culture are produced from a plant part selected from the groupconsisting of protoplasts, embryos, meristematic cells, callus, pollen,ovules, flowers, seeds, leaves, roots, root tips, anthers, stems,petioles, head, axillary buds, cotyledons and hypocotyls. In someembodiments, the plant part includes protoplasts produced from a plantgrown from the deposited lettuce cultivar DESERT EAGLE seed.

In some embodiments, the present invention teaches a lettuce plantregenerated from the tissue culture from a plant grown from thedeposited lettuce cultivar DESERT EAGLE seed, said plant having thecharacteristics of lettuce cultivar DESERT EAGLE, wherein arepresentative sample of seed of said lettuce cultivar DESERT EAGLE isdeposited under NCIMB No. 42806.

In some embodiments, the present invention teaches a lettuce headproduced from plants grown from the deposited lettuce cultivar DESERTEAGLE seed.

In some embodiments, methods of producing said lettuce head comprise a)growing the lettuce plant from deposited lettuce cultivar DESERT EAGLEseed to produce a lettuce head, and b) harvesting said lettuce head. Insome embodiments, the present invention also teaches a lettuce headproduced by the method of producing lettuce head as described above.

In some embodiments, the present invention teaches methods for producinga lettuce seed comprising crossing a first parent lettuce plant with asecond parent lettuce plant and harvesting the resultant lettuce seed,wherein said first parent lettuce plant and/or second parent lettuceplant is the lettuce plant produced from the deposited lettuce cultivarDESERT EAGLE seed, or a lettuce plant having all of the characteristicsof lettuce cultivar DESERT EAGLE as listed in Table 1 of thisapplication.

In some embodiments, the present invention teaches methods for producinga lettuce seed comprising self-pollinating the lettuce plant grown fromthe deposited lettuce cultivar DESERT EAGLE seed and harvesting theresultant lettuce seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the lettuce plant grown from the depositedlettuce cultivar DESERT EAGLE seed, said method comprising a) collectingpart of a plant grown from the deposited lettuce cultivar DESERT EAGLEseed and b) regenerating a plant from said part.

In some embodiments, the method further comprises harvesting a head fromsaid vegetatively propagated plant.

In some embodiments, the present invention teaches the plant, the headand leaves thereof of plants vegetatively propagated from plant parts ofplants grown from the deposited lettuce cultivar DESERT EAGLE seed.

In some embodiments, the present invention teaches methods of producinga lettuce plant derived from the lettuce cultivar DESERT EAGLE. In someembodiments, the methods comprise the steps of: (a) self-pollinating theplant grown from the deposited lettuce cultivar DESERT EAGLE seed atleast once to produce a progeny plant derived from lettuce cultivarDESERT EAGLE; (b) crossing the progeny plant derived from lettucecultivar DESERT EAGLE with itself or a second lettuce plant to produce aseed of a progeny plant of a subsequent generation and; (c) growing theprogeny plant of the subsequent generation from the seed and crossingthe progeny plant of the subsequent generation with itself or a secondlettuce plant to produce a lettuce plant derived from the lettucecultivar DESERT EAGLE. In some embodiments said method further comprisesthe step of: (d) repeating steps b) and/or step c) for at least 1, 2, 3,4, 5, 6, 7 more generation to produce a lettuce plant derived from thelettuce cultivar DESERT EAGLE.

In some embodiments, the present invention teaches methods of producinga lettuce plant derived from the lettuce cultivar DESERT EAGLE, themethods comprising the steps of: (a) crossing the plant grown from thedeposited lettuce cultivar DESERT EAGLE seed with a second lettuce plantto produce a progeny plant derived from the lettuce cultivar DESERTEAGLE; In some embodiments, the method further comprises (b) crossingthe progeny plant derived from the lettuce cultivar DESERT EAGLE withitself or a second lettuce plant to produce a seed of a progeny plant ofa subsequent generation and; (c) growing the progeny plant of thesubsequent generation from the seed and crossing the progeny plant ofthe subsequent generation with itself or a second lettuce plant toproduce a lettuce plant derived from the lettuce cultivar DESERT EAGLE.In some embodiments said method further comprises the steps of: (d)repeating steps b) and/or c) for at least 1, 2, 3, 4, 5, 6, 7 moregeneration to produce a lettuce plant derived from the lettuce cultivarDESERT EAGLE.

In some embodiments, the present invention teaches plants grown from thedeposited lettuce cultivar DESERT EAGLE seed wherein said plantscomprise at least one single locus conversion. In some embodiments saidsingle locus conversion confers said plant with a trait selected fromthe group consisting of male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, heat tolerance, delayed senescence, improved ripeningcontrol, long shelf life, and improved salt tolerance when compared to asuitable check plant. In some embodiments, the check plant is a lettuceDESERT EAGLE cultivar not having said single locus conversion. In someembodiments, the at least one single locus conversion is an artificiallymutated gene or a gene or nucleotide sequence modified through the useof New Breeding Techniques.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Big Vein. Big vein is a disease of lettuce caused by Mirafiori lettuceBig Vein Virus (MiLBVV, genus Ophiovirus) which is transmitted by thefungus Olpidium virulentus, with vein clearing and leaf shrinkageresulting in plants of poor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants

Core length. The core length is the length of the internal lettuce stemmeasured from the base of the cut and trimmed head to the tip of thestem.

Core diameter: Core diameter is the diameter of the internal leaf stemmeasured at the base of the head.

Corky root. Corky root is a disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Cupping. In romaine lettuce, cupping is the process by which the romaineform a heart. Leaves of similar size are formed in the center of thehead and then, the tops of the leaves fold downwards slightly to form aheart.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date

Frame diameter: For frame diameter in case of romaine lettuce, themeasurement is taken from the outer most leaf tip horizontally to theouter most leaf tip. In case of icebergs, frame diameter is measuredwith the outer leaves intact

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem in case ofromaine lettuce. In case of icebergs, head diameter is measured afterremoving the outer leaves (just the round head).

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Immunity to disease(s) and or insect(s). A lettuce plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Intermediate/Moderate resistance to disease(s) and or insect(s). Alettuce plant that restricts the growth and development of specificdisease(s) and or insect(s), but may exhibit a greater range of symptomsor damage compared to high/standard resistant plants. Intermediateresistant plants will usually show less severe symptoms or damage thansusceptible plant varieties when grown under similar environmentalconditions and/or specific disease(s) and or insect(s) pressure, but mayhave heavy damage under heavy pressure. Intermediate resistant lettuceplants are not immune to the disease(s) and or insect(s).

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Lettuce Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe lettuce at harvest.

Maturity (Date). Maturity refers to the stage when plants are of fullsize or optimum weight, and in marketable form or shape to be ofcommercial or economic value. In leaf types they range from 50-75 daysfrom time of seeding, depending upon the season of the year. In othertypes, they range from 65-105 days from time of seeding, depending uponthe season of the year

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

New Breeding Techniques: New breeding techniques are said of various newtechnologies developed and/or used to create new characteristics inplants through genetic variation, the aim being targeted mutagenesis,targeted introduction of new genes or gene silencing (RdDM). Example ofsuch new breeding techniques are targeted sequence changes facilitatedthru the use of Zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2 andZFN-3, see U.S. Pat. No. 9,145,565, incorporated by reference in itsentirety), Oligonucleotide directed mutagenesis (ODM), Cisgenesis andintragenesis, RNA-dependent DNA methylation (RdDM, which does notnecessarily change nucleotide sequence but can change the biologicalactivity of the sequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration (agro-infiltration “sensu stricto”, agro-inoculation,floral dip), Transcription Activator-Like Effector Nucleases (TALEN5,see U.S. Pat. Nos. 8,586,363 and 9,181,535, incorporated by reference intheir entireties), the CRISPR/Cas system (see U.S. Pat. Nos. 8,697,359;8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308;8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are allhereby incorporated by reference), engineered meganuclease re-engineeredhoming endonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics). A complete description ofeach of these techniques can be found in the report made by the JointResearch Center (JRC) Institute for Prospective Technological Studies ofthe European Commission in 2011 and titled “New plant breedingtechniques—State-of-the-art and prospects for commercial development”,which is incorporated by reference in its entirety.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant Cell. Plant cell, as used herein includes plant cells whetherisolated, in tissue culture or incorporated in a plant or plant part.

Plant Part. As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, heads, rootstock, scions, stems, roots, anthers, pistils, roottips, leaves, meristematic cells, axillary buds, hypocotyls cotyledons,ovaries, seed coat endosperm and the like.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. The ratio is the head height divided bythe head diameter and is an indication of the head shape; <1 isflattened, 1=round, and >1 is pointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A lettuce plant thatrestricts highly the growth and development of specific disease(s) andor insect(s) under normal disease(s) and or insect(s) attack pressurewhen compared to susceptible plants. These lettuce plants can exhibitsome symptoms or damage under heavy disease(s) and or insect(s)pressure. Resistant lettuce plants are not immune to the disease(s) andor insect(s).

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Hort. Society Enterprise Ltd. RHS Garden; Wisley,Woking, Surrey GU236QB, UK.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering. A single gene converted plant canalso be referred to a plant obtained though mutagenesis or through theuse of some new breeding techniques, whereas the single gene convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to the single geneor nucleotide sequence mutated or engineered through the new breedingtechniques.

Susceptible to disease(s) and or insect(s). A lettuce plant that issusceptible to disease(s) and or insect(s) is defined as a lettuce plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tip burn. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium

Tolerance to abiotic stresses. A lettuce plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Lettuce Plants

Most cultivated forms of lettuce belong to the highly polymorphicspecies Lactuca sativa which is grown for its edible head and leaves. Asa crop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield. Lettuce is theworld's most popular salad. In the United States, the principal growingregions are California and Arizona which produce approximately 329,000acres out of a total annual acreage of more than 333,000 acres (USDA,2005). Fresh lettuce is available in the United States year-roundalthough the greatest supply is from May through October. For plantingpurposes, the lettuce season is typically divided into three categories,early, mid and late, with the coastal areas planting from January toAugust, and the desert regions from August to December. Lettuce isconsumed nearly exclusively as fresh, raw product, and occasionally as acooked vegetable. Baby leaf or spring mix lettuce is an increasinglypopular crop as worldwide baby leaf lettuce consumption continues toincrease. Spring mix lettuce refers to lettuce that is grown in highconcentrations and harvested at a very young or ‘baby leaf’ stage,typically 30 to 45 days after planting. The plantings are often done onwider 80 to 84 inch beds and often contain up to one million plants peracre. Compared to iceberg or romaine plantings, where they are typicallyharvested 60 to 100 days after planting, with a population of roughly25,000 to 30,000 plants per acre. Spring mix plantings often includemultiple types of lettuces, all harvested when the leaves are young andtender. These plantings can include green romaine, red romaine, darklolla rossa, tango, green leaf, and red leaf types. Spring mix fieldsare most often harvested mechanically and the harvested leaves arepacked in plastic totes, where they are transported to a processingfacility where they are washed, processed and mixed according to thesalad recipe.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositaefamily). Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are several morphological types of lettuce. TheCrisphead group includes the Iceberg and Batavian types. Iceberg lettucehas a large, firm head with a crisp texture and a white or creamy yellowinterior. Batavian lettuce predates Iceberg lettuce and has a smallerand less firm head. The Butterhead group has a small, soft head with analmost oily texture. Romaine lettuce, also known as Cos lettuce, haselongated upright leaves forming a loose, loaf-shaped head and the outerleaves are usually dark green. Leaf lettuce comes in many varieties,none of which form a head. There are three types of lettuce which areseldom seen in the United States: Latin lettuce, which looks like across between Romaine and Butterhead; Stem lettuce, which has long,narrow leaves and thick, edible stems; and Oilseed lettuce, which is aprimitive type of lettuce grown for its large seeds that are pressed toobtain oil.

Lactuca sativa is normally a simple diploid species with nine pairs ofchromosomes (2N=18). However, haploidy and polyploidy lettuce plants arealso part of the present invention. Lettuce is an obligateself-pollinating species which means that pollen is shed before stigmaemergence, assuring 100% self-fertilization. Since each lettuce floweris an aggregate of about 10-20 individual florets (typical of theCompositae family), manual removal of the anther tubes containing thepollen is tedious. As a result, a modified method of misting to wash offthe pollen prior to fertilization is needed to assure crossing orhybridization. Flowers to be used for crossings are selected about 60-90minutes after sunrise. Selection criteria include plants with openflowers, where the stigma has emerged and pollen is visibly attached toa single stigma (there are about 10-20 stigma). Pollen grains are washedoff using 3-4 pumps of water from a spray bottle and with enoughpressure to dislodge the pollen grains without damaging the style.Excess water is then dried off using clean paper towels and about 30minutes later, the styles spring back up and the two lobes of the stigmaare visibly open in a “V” shape. Pollen from another variety or donorparent is then introduced by gently rubbing the stigma and style of thedonor parent to the maternal parent. Most pertinent informationincluding dates and pedigree are then secured to the flowers using tags.

Hybrid vigor has been documented in lettuce and hybrids will be gainingmore and more popularity amongst farmers with uniformity of plantcharacteristics.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In lettuce, these important traits may include increased head size andweight, higher seed yield, improved color, resistance to diseases andinsects, tolerance to drought and heat, better post-harvest shelf-lifeof the leaves, better standing ability in the field, better uniformity,and better agronomic quality.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to fungi such as Bremia lactucae, Fusarium oxysporum,Sclerotinia minor or sclerotorum, Botrytis cinerea, Rhizictonia solani,Microdochium panattonianum, Verticiulium dahliae, Erysiphe chicocearumor Pithium tracheiphilum, virus, such as LMV (lettuce mosaic virus),TSWV (tomato potted wilt virus), “Big vein” (composed of LBW (lettucebig vein virus) and MILV (miratiori lettuce virus)), TBSV (tomato bushystunt virus), LNSV (lettuce necrotic stunt virus), TuMV (turnip mosaicvirus), CMV (cucumber mosaic virus) or BWYV (beet western yellowsvirus), bacteria such as Pseudomonas, Xanthomonas or Rhizomonas.Improved resistance to insect pests is another desirable trait that maybe incorporated into new lettuce plants developed by this invention.Insect pests affecting the various species of lettuce include Nasonoviaribisnigri, Myzus persicae, Macrosiphum euphorbia, Nematodespratylenchus or meloidogyne, leafminers: Liriomyza huidobrensis orPemphigus busarius.

Other desirable traits include traits related to improved lettuce plantsand parts thereof. A non-limiting list of lettuce phenotypes used duringbreeding selection includes:

-   -   Tomato Bushy Stunt (TBSV) resistance or tolerance. TBSV is a        viral disease which causes stunting of growth, leaf mottling,        and deformed or absent heads. When associated with Lettuce        Necrotic Stunt Virus (LNSV), another soil born virus, Tomato        Bushy Stunt leads to the disease known as Dieback (Simko et al.,        2010. HortScience 45(2): 670-672), resulting in mottling,        yellowing, and necrosis of older leaves, stunting of the plant,        and eventually death    -   Tip Burn tolerance. Tip burn tolerance is a tolerance to an        abiotic disorder caused by calcium deficiency in growing tissues        and resulting in the browning, up to black color, of the margins        of young, maturing leaves in head and leaf lettuces. The brown        area may be limited to a few small spots at or near the leaf        margin, or the entire edge of the leaf may be affected. The term        tip burn is usually used to refer to the browning in the        internal leaves of the plant. Tip burn is also caused by        environmental conditions that reduce transpiration such as foggy        conditions and soil water stress (source: UC Pest Management        Guidelines)    -   Fringe burn tolerance. Fringe burn tolerance is tolerance to        brown discoloration on the outer edge of the lettuce leaf.        Fringe burn may be limited to a few spots or cover the entire        edge of the leaf. The term Fringe burn is usually used to refer        to browning on the external leaves of the plant.        Lettuce Breeding

The goal of lettuce breeding is to develop new, unique and superiorlettuce cultivar and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Another method used to developnew, unique and superior lettuce cultivar occurs when the breederselects and crosses two or more parental lines followed by haploidinduction and chromosome doubling that result in the development ofdihaploid cultivars. The breeder can theoretically generate billions ofdifferent genetic combinations via crossing, selfing and mutations andthe same is true for the utilization of the dihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting cultivars he develops, exceptpossibly in a very gross and general fashion. This unpredictabilityresults in the expenditure of large research monies to develop superiornew lettuce cultivars.

The development of commercial lettuce cultivar requires the developmentand selection of lettuce plants, the crossing of these plants, and theevaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes or through the dihaploidbreeding method followed by the selection of desired phenotypes. The newcultivars are evaluated to determine which have commercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease of new cultivars. Similarly, the development of new cultivarsthrough the dihaploid system requires the selection of the cultivarsfollowed by two to five years of testing in replicated plots.

ii Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

When the term lettuce cultivar is used in the context of the presentinvention, this also includes any lettuce cultivar plant where one ormore desired trait has been introduced through backcrossing methods,whether such trait is a naturally occurring one, a mutant a gene or anucleotide sequence modified by the use of New Breeding Techniques.Backcrossing methods can be used with the present invention to improveor introduce one or more characteristic into the lettuce cultivar of thepresent invention. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent,i.e., backcrossing one, two, three, four, five, six, seven, eight, nine,or more times to the recurrent parent. The parental lettuce cultivarplant which contributes the gene or the genes for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental lettuce cultivar to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol.

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It has to be noted that some,one, two, three or more, self-pollination and growing of populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic in corn, require selfing the progeny to determine whichplant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridlettuce plant according to the invention but that can be improved bybackcrossing techniques. Single gene traits may or may not betransgenic. Examples of these traits include but are not limited to,male sterility (such as a PR glucanase gene or the ms1, ms2, ms3, ms4,ms5, ms7 genes), herbicide resistance (such as bar or PAT genes), Manysingle gene traits have been identified that are not regularly selectedfor in the development of a new line but that can be improved bybackcrossing techniques. Single gene traits may or may not betransgenic. An example of a gene controlling resistance to the lettuceleaf aphid Nasonovia ribisnigri (Nr gene) can be found in Van der Arendand Schijndel in Breeding for Resistance to insects and Mites, IOBC wprsBulletin 22(10), 35-43 (1999). Other traits for resistance or toleranceto an infection by a virus, a bacterium, an insect or a fungus, might beobtained from the genes for resistance to Bremia Dm10, R17, Dm5, Dm8,R36, R37 (genes located on cluster 1 of Lactuca sativa), Dm1, Dm2, Dm3,Dm6, Dm14, Dm15, Dm16, Dm18 (genes located on cluster 2 of Lactucasativa), Dm4, Dm7, Dm11, R38 (genes located on cluster 4 of Lactucasativa); or the Tu gene for resistance to TuMV located on cluster 1; orfrom the genes mol.1 and mol.2 for resistance to LMV located on cluster4. Clusters 1, 2 and 4 cited above have been defined by Michelmore R. W.(Plant Pathol, 1987, vol. 36, no 4: 499-514 [4], Theor. Appl. Genet.,1993, vol. 85, No 8: 985-993. These genes are generally inheritedthrough the nucleus. Some other single gene traits are described in U.S.Pat. Nos. 5,777,196, 5,948,957, and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc, Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly theadaptation, yielding ability and quality characteristics of therecurrent parent but superior to that parent in the particularcharacteristic(s) for which the improvement program was undertaken.Therefore, this method provides the plant breeder with a high degree ofgenetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,colour characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii Single-Seed Descent and Multiple Seed Procedures

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or moreflower containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each flower by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

iii Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,maize and sugar beets, herbage grasses, legumes such as alfalfa andclover, and tropical tree crops such as cacao, coconuts, oil palm andsome rubber, depends essentially upon changing gene-frequencies towardsfixation of favorable alleles while maintaining a high (but far frommaximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagable by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagable as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortoperosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial lettuce seed is produced by insect pollination seeU.S. Pat. No. 8,716,551 which is specifically hereby incorporated byreference.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337): 1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs). Markers linked to thephenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Insome embodiments the donor or recipient female parent and the donor orrecipient male parent line are planted in the same greenhouse. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. Pollination is started when thefemale parent flower is ready to be fertilized. Female flower buds thatare ready to open in the following days are identified, covered withpaper cups or small paper bags that prevent bee or any other insect fromvisiting the female flowers, and marked with any kind of material thatcan be easily seen the next morning. The male flowers of the male parentare collected in the early morning before they are open and visited bypollinating insects. The covered female flowers of the female parent,which have opened, are un-covered and pollinated with the collectedfresh male flowers of the male parent, starting as soon as the maleflower sheds pollen. The pollinated female flowers are again coveredafter pollination to prevent bees and any other insects visit. Thepollinated female flowers are also marked. The marked flowers areharvested. In some embodiments, the male pollen used for fertilizationhas been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Insects are placed inthe field or greenhouses for transfer of pollen from the male parent tothe female flowers of the female parent.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant cultivars. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. A “bubble” forms at the mismatch of thetwo DNA strands (the induced mutation in TILLING® or the naturalmutation or SNP in EcoTILLING), which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

viii Mutation Breeding

Mutation breeding is another method of introducing new traits intolettuce plants. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means or mutating agents including temperature, long-term seedstorage, tissue culture conditions, radiation (such as X-rays, Gammarays, neutrons, Beta radiation, or ultraviolet radiation), chemicalmutagens (such as base analogs like 5-bromo-uracil), antibiotics,alkylating agents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in W. R. Fehr, 1993, Principles ofCultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intolettuce varieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple back-crossings is to produce haploids and then doublethe chromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol109, pg 4227-4232; Zhang et al., 2008 Plant Cell Rep. Dec 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg 63-72; Doubled Haploid Production in Crop Plants2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform cultivars and is especiallydesirable as an alternative to sexual inbreeding of longer-generationcrops. By producing doubled haploid progeny, the number of possible genecombinations for inherited traits is more manageable. Thus, an efficientdoubled haploid technology can significantly reduce the time and thecost of inbred and cultivar development.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryo's fromcrosses wherein plants fail to produce viable seed. In this process, thefertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In vitro culture of higher plants,Springer, ISBN 079235267x, 9780792352679, which is incorporated hereinby reference in its entirety).

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, height, weight, color, taste, smell, changes in theproduction of one or more compounds by the plant (including for example,metabolites, proteins, drugs, carbohydrates, oils, and any othercompounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil) or biomass production; effects on plant growththat results in an increased seed yield for a crop; effects on plantgrowth which result in an increased head yield; effects on plant growththat lead to an increased resistance or tolerance disease includingfungal, viral or bacterial diseases or to pests such as insects, mitesor nematodes in which damage is measured by decreased foliar symptomssuch as the incidence of bacterial or fungal lesions, or area of damagedfoliage or reduction in the numbers of nematode cysts or galls on plantroots, or improvements in plant yield in the presence of such plantpests and diseases; effects on plant growth that lead to increasedmetabolite yields; effects on plant growth that lead to improvedaesthetic appeal which may be particularly important in plants grown fortheir form, color or taste, for example the color intensity of lettuceleaves, or the taste of said leaves.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Whole Transcriptome Shotgun Sequencing),qRTPCR (quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, RIA, immune labeling, immunosorbent electron microscopy(ISEM), and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene or a desirable trait (e.g., a co-segregating nucleicacid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the cDNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cations concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing agarose gel electrophoresis and ethidium bromide (or other nucleicacid staining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. This, combinedwith the fact that ethidium bromide has low sensitivity, yields resultsthat are not always reliable. Moreover, there is an increasedcross-contamination risk of the samples since detection of the PCRproduct requires the post-amplification processing of the samples.Furthermore, the specificity of the assay is mainly determined by theprimers, which can give false-positive results. However, the mostimportant issue concerning conventional RT-PCR is the fact that it is asemi or even a low quantitative technique, where the amplicon can bevisualized only after the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general non specific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al., 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg169-200; Mardis 2008 Genomics and Human Genetics vol 9 pg 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line or cultivarhaving certain favorite traits such for commercial production. In oneembodiment, the elite line may contain other genes that also impart saidline with the desired phenotype. When crossed together, the donor andrecipient plant may create a progeny plant with combined desirable lociwhich may provide quantitatively additive effect of a particularcharacteristic. In that case, QTL mapping can be involved to facilitatethe breeding process.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait—these QTLs are often found ondifferent chromosomes. Knowing the number of QTLs that explainsvariation in the phenotypic trait tells about the genetic architectureof a trait. It may tell that a trait is controlled by many genes ofsmall effect, or by a few genes of large effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing a gene that is associated with the traitbeing assayed or measured. They are shown as intervals across achromosome, where the probability of association is plotted for eachmarker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, and dothose loci interact. This can provide information on how the phenotypemay be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency corresponds to a low distance between markers ona chromosome. Comparing all recombination frequencies will result in themost logical order of the molecular markers on the chromosomes. Thismost logical order can be depicted in a linkage map (Paterson, 1996,Genome Mapping in Plants. R. G. Landes, Austin.). A group of adjacent orcontiguous markers on the linkage map that is associated to a reduceddisease incidence and/or a reduced lesion growth rate pinpoints theposition of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to make incorporate thedesirable train into progeny plants by transferring and/or breedingmethods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred as near isogenic lines (NILs), introgression lines (ILs),backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises of a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of lettuce can be usedfor the in vitro regeneration of lettuce plants. Tissues cultures ofvarious tissues of lettuce and regeneration of plants therefrom are wellknown and published. For example, reference may be had to Teng et al.,HortScience, 27: 9, 1030-1032 (1992), Teng et al., HortScience. 28: 6,669-671 (1993), Zhang et al., Journal of Genetics and Breeding, 46: 3,287-290 (1992), Webb et al., Plant Cell Tissue and Organ Culture, 38: 1,77-79 (1994), Curtis et al., Journal of Experimental Botany, 45: 279,1441-1449 (1994), Nagata et al., Journal for the American Society forHorticultural Science, 125: 6, 669-672 (2000). It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of lettuce cultivarDESERT EAGLE.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1—Development of Lettuce Cultivar DESERT EAGLE

The lettuce cultivar DESERT EAGLE is an iceberg variety with large frameand big head's size. It produces nice round heads with very smooth buttappearance. It is a high yield potential iceberg variety and has showngood adaptability to production areas of Yuma from warm to coldtransition weather condition. Variety Desert Eagle has no resistance toany Bremia races.

Breeding History:

DESERT EAGLE has superior characteristics and was developed from thecross between the commercial varieties Mohawk, Early Queen and Tiber.The original cross was made in the summer of the first year in agreenhouse at Shamrock Seed Company research station in Gilroy, Calif.The F1 plants were grown in greenhouse at Shamrock Seed Company researchstation in Gilroy, Calif. in the second year and F1 plants were allowedto self and F2 seeds were collected from each of the F1 plantsseparately. In September of the third year, the F2 seeds were planted inYuma, Ariz., and three individual plants were selected and allowed toself-pollinate. The F3 seeds were collected individually from each F2plant. In September of the fourth year, a total of three F3 familieswere planted and three individual plants were selected from two F3family lines. All individual plants selected were allowed to self andthe F4 seeds collected individually from each F3 plant. In September ofyear five, one F4 lines were planted and two individual plants wereselected to obtain F5 seeds. In September of year six, in a Yuma trial,two F5 lines were planted and three individual plants were selected fromthese F5 lines. All three plants were allowed to self and seeds werecollected individually from each plant to obtain F6 seeds. In Septemberof year seven, F6 lines were planted and seven individual plants wereselected. All plants were allowed to self and F7 seeds were collectedindividually from each F6 plant. In year eight of development, F7 seedswere planted in Gilroy, Calif. in seed increase cage. All plants wereharvested in bulk to create romaine lettuce variety DESERT EAGLE. Someof the criteria used to select the lettuce cultivar DESERT EAGLE invarious generations include: global frame and head size, head shape andplant architecture, strength and texture of leaves, diseasesresistances, weight and yield, maturity, ease to harvest, processability, resistance to internal tip burn (necrosis), resistance tobolting, resistance to head cracking under over mature conditions, seedqualities.

DESERT EAGLE is an iceberg type similar to cultivar Midway and Javalinabut has numerous differences.

The lettuce cultivar DESERT EAGLE has shown uniformity and stability forthe traits, within the limits of environmental influence for the traitsas described in the following Variety Descriptive Information. Novariant traits have been observed or are expected for agronomicalimportant traits in lettuce cultivar DESERT EAGLE.

Lettuce cultivar DESERT EAGLE has the following morphologic and othercharacteristics, (based primarily on data collected in California, allexperiments done under the direct supervision of the applicant).

TABLE 1 Variety Description Information Plant: Type: Iceberg Seed:Color: Black Cotyledon to Fourth Leaf Stage: Shape of cotyledon: BroadCotyledon leaf index 2.4 Shape of fourth leaf: Elongated Fourth leafindex 2.1 Apical Margin: Entire Basal Margin: Entire Undulation: SlightGreen Color: Medium Green Anthocyanin Distribution: Absent AnthocyaninConcentration: N/A Rolling: Absent Cupping: Uncupped Reflexing: NoneHarvest-Mature Out Leaf, Head, Core: Margin Incision Depth: ModerateMargin Indentation: Shallowly Dentate Undulation of the Apical Margin:Moderate Green Color: Dark Green Anthocyanin Distribution: AbsentAnthocyanin Concentration: N/A Anthocyanin Size: N/A Glossiness:Moderate Blistering: Moderate Leaf Thickness: Thick Trichomes: Absent(Smooth) Head Shape: Flattened Head Size Glass: Medium to Large Head PerCarton: 24 Head Firmness: Firm Butt Shape: Flat Butt Midrib: ModeratelyRaised Maturity (No. of Days of First Water date to Harvest): Fall(Desert South West): 78 to 105 Outer Leaf Length (cm): 25.9 Out LeafWidth (cm): 28.3 Leaf index 0.92 Leaf area (cm2) 742.63 Head Weight (g):759.3 Head Length (cm): 13.7 Head Diameter (cm): 13.8 Core Length (mm):45.6 Diameter (mm): 34.8 Adaptation: Primary Regions of Adaptation(tested and proven adapted): Yuma (AZ, USA) Season: Warm to coldtransition for Yuma Soil Type: Adapted to most soil types Diseases:Tomato Bushy stunt virus Resistant Downy Mildew: No resistanceSclerotinia Rot: Not tested Nasonovia ribisnigri: susceptibleSusceptible Physiological/Stress: Tipburn: Good level of toleranceBolting: Good level of tolerance

Example 2—Field Trials Characteristics of Lettuce Cultivar DESERT EAGLE

In the following tables, several traits and characteristics of lettucecultivar DESERT EAGLE are compared to Midway and Javalina varieties. Thedata was collected from various field locations in the United States.The field tests are experimental trials and have been made undersupervision of the applicant.

Table 2 Presents the Length of Cotyledon Leaf Measured in mm at 20 DaysOld Seedlings

Desert Eagle Javalina Midway Cotyledon length (mm) 24 24 24 13 20 24 2621 22 23 23 24 9 17 28 11 13 23 7 25 23 22 25 21 14 19 25 13 17 20Anova: Single Factor SUMMARY Groups Count Sum Average Variance DesertEagle 10 162 16.2 47.29 Javalina 10 204 20.4 15.82 Midway 10 234 23.44.93 ANOVA P- Source of Variation SS df MS F value F crit Between Groups261.6 2 130.8 5.7668 0.0082 3.3541 Within Groups 612.4 27 22.68 Total874 29 Cotyledon length (mm) summary: ANOVA shows a significantdifference (p < .01) in the length of cotyledon leaf measured in mm on20 day old seedlings.Table 3 Presents the Width of Cotyledon Leaf Measured in mm at 20 DaysOld Seedlings

Desert Eagle Javalina Midway Cotyledon Width (mm) 9 8 9 4 11 11 9 10 8 68 10 6 8 11 8 11 10 5 10 10 9 9 9 7 10 9 6 9 9 Anova: Single FactorSUMMARY Groups Count Sum Average Variance Desert Eagle 10 69 6.9 3.21Javalina 10 94 9.4 1.38 Midway 10 96 9.6 0.93 ANOVA P- Source ofVariation SS df MS F value F crit Between Groups 45.27 2 22.63 12.29580.0002 3.3541 Within Groups 49.7 27 1.84 Total 94.97 29 Cotyledon width(mm) summary: ANOVA shows a significant difference (p < .001) in thewidth of cotyledon leaf measured in mm on 20 day old seedlings.Table 4 Presents the Cotyledon Index (Calculated by Dividing theCotyledon Leaf Length by the Cotyledon Leaf Width)

Desert Eagle Javalina Midway 2.7 3.0 2.7 3.3 1.8 2.2 2.9 2.1 2.8 3.8 2.92.4 1.5 2.1 2.5 1.4 1.2 2.3 1.4 2.5 2.3 2.4 2.8 2.3 2.0 1.9 2.8 2.2 1.92.2 Anova: Single Factor SUMMARY Groups Count Sum Average VarianceDesert Eagle 10 23.53 2.35 0.68 Javalina 10 22.17 2.22 0.32 Midway 1024.48 2.45 0.05 ANOVA P- Source of Variation SS df MS F value F critBetween Groups 0.27 2 0.13 0.3840 0.6848 3.3541 Within Groups 9.48 270.35 Total 9.75 29 Cotyledon leaf index summary: ANOVA shows nosignificant difference (p < .05) in the cotyledon leaf index measured inmm on 20 day old seedlings.Table 5 Presents the Length of the 4th True Leaf was Measured in mm on20 Days Old Seedlings.

Desert Eagle Javalina Midway 4th Leaf Length (mm) 42.0 50.0 35.0 41.057.0 53.0 52.0 55.0 50.0 36.0 42.0 49.0 50.0 46.0 51.0 54.0 48.0 49.039.0 48.0 38.0 46.0 49.0 46.0 26.0 41.0 55.0 41.0 55.0 53.0 Anova:Single Factor SUMMARY Groups Count Sum Average Variance Desert Eagle 10427 42.7 69.12 Javalina 10 491 49.1 28.99 Midway 10 479 47.9 42.99 ANOVAP- Source of Variation SS df MS F value F crit Between Groups 231.47 2115.73 2.4607 0.1043 3.3541 Within Groups 1269.90 27 47.03 Total 1501.3729 4th leaf length (mm) summary: ANOVA shows no significant difference(p < .05) in the length of 4th leaf measured in mm on 20 day oldseedlings.Table 6 Presents the Width of 4th True Leaf Measured in mm on 20 DaysOld Seedlings

Desert Eagle Javalina Midway 4th Leaf Width (mm) 16.0 20 16 18.0 14 1821.0 16 21 20.0 20 19 27.5 18 19 26.0 17 18 18.0 20 19 23.0 16 17 19.016 18 15.0 24 20 Anova: Single Factor SUMMARY Groups Count Sum AverageVariance Desert Eagle 10 203.5 20.35 16.78 Javalina 10 181 18.1 8.54Midway 10 185 18.5 2.06 ANOVA P- Source of Variation SS df MS F value Fcrit Between Groups 28.82 2 14.41 1.5787 0.2247 3.3541 Within Groups246.43 27 9.13 Total 275.24 29 4th leaf width (mm) summary: ANOVA showsno significant difference (p < .05) in the width of 4th leaf measured inmm on 20 day old seedlings.Table 7 Presents the 4th Leaf Index (Calculated by Dividing the 4th LeafLength by the 4th leaf width measured on 20 days old seedlings)

Desert Eagle Javalina Midway 2.6 2.5 2.2 2.3 4.1 2.9 2.5 3.4 2.4 1.8 2.12.6 1.8 2.6 2.7 2.1 2.8 2.7 2.2 2.4 2.0 2.0 3.1 2.7 1.4 2.6 3.1 2.7 2.32.7 Anova: Single Factor SUMMARY Groups Count Sum Average VarianceDesert Eagle 10 21.34 2.13 0.17 Javalina 10 27.80 2.78 0.36 Midway 1025.91 2.59 0.10 ANOVA P- Source of Variation SS df MS F value F critBetween Groups 2.21 2 1.10 5.2266 0.0121 3.3541 Within Groups 5.70 270.21 Total 7.91 29 4th leaf index summary: ANOVA shows a significantdifference (p < .05) in the 4th leaf index measured in mm on 20 day oldseedlings.Table 8 Presents the Plant Weight (g) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert938 550 575 840 760 882 645 Eagle 855 670 655 721 797 687 650 950 710580 970 805 1033 722 458 780 630 900 916 1047 625 553 800 455 1450 7911065 650 371 890 485 675 706 882 735 940 750 635 820 838 1053 583 486650 545 600 778 779 880 840 750 490 705 950 921 815 500 850 720 675 9471031 760 Javalina 948 738 815 940 1093 613 610 934 770 985 700 1048 931680 702 800 580 800 886 831 595 508 850 500 500 1041 623 653 721 750 935735 1033 892 550 885 650 870 860 972 897 492 533 580 655 804 984 533 525895 590 690 630 604 780 750 1198 500 670 1200 1195 855 660 907 600 770650 856 582 760 Midway 1134 936 755 1066 757 725 855 1349 1100 645 1095926 806 855 954 795 445 645 982 915 1043 1008 805 715 990 957 507 604824 950 370 1760 1197 869 960 970 990 665 1109 1284 836 989 927 1100 6051265 839 731 923 937 1115 660 855 951 672 772 787 1050 410 1455 947 720770 978 980 330 1160 1047 572 785 Anova: Two-Factor With ReplicationSUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Total DesertEagle Count 10 10 10 10 10 10 10 70 Sum 6891 7400 5770 8356 8288 93807065 53150 Average 689.1 740 577 835.6 828.8 938 706.5 759.29 Variance54821.21 10000.00 7184.44 59693.60 6861.07 17083.56 8650.06 33590.12Javalina Count 10 10 10 10 10 10 10 70 Sum 8231 6828 7470 7819 9712 75376275 53872 Average 823.1 682.8 747 781.9 971.2 753.7 627.5 769.6Variance 43669.43 13042.84 24312.22 37284.99 26115.73 22566.01 8140.0633299.20 Midway Count 10 10 10 10 10 10 10 70 Sum 9868 9821 5600 114009887 7353 8556 62485 Average 986.8 982.1 560 1140 988.7 735.3 855.6892.64 Variance 25246.84 13296.32 24116.67 95206.89 24247.34 16495.5716773.38 60566.03 ANOVA Source of Variation SS df MS F P-value F critVariety  770701.32 2 385350.66 14.5859 <.0001 3.0437 Location 1989092.516 331515.42 12.5482 <.0001 2.1468 Interaction 1812052.54 12 151004.385.7157 <.0001 1.8037 Within 4993274.10 189  26419.44 Total 9565120.48209 ANOVA shows significant differences (p < .0001) in plant wt. (g)between varieties, location, and variety*location interaction at harvestmaturity stage.Table 9 Presents the Head Length (cm) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert14 15 13 14 12.5 16 12 Eagle 14 12 13 12.5 13 15 14 14 13 14 14 13.5 1413 12 14 14 13.5 12.5 13 13 12 15 11.5 15.5 13 15 14 14 16 13 13.5 13 1413 16 14 12 13 12.5 15 14 12 16 15 12 13.5 15 14 15.5 13 13 13 13 14 1513.5 13 14 13.5 13 14 14 Javalina 13 15 12 15 14 14 12 14 13 13 13 15 1311 13 12 12 14 15 13 13 10 14 13 11 14 13 12 16 15 13 12.5 14 13 13 12.514 14 14 12 13 13.5 12 13 13 13 14 13 13 14 13 13 12.5 14 12 12 14 12 1415.5 13 12 12 15.5 13 11.5 12 11 14 12 Midway 17 16 15 15 13 13 14 16 1616 14.5 15 13 14 15 16 14 13.5 15 14 13 13 15 17 14 15 14 14 14 16 1316.5 15 14 16 15.5 16 14 15 14 14 15 15 16 14 16.5 13 14 14.5 15 15 1614 12.5 14 13 14.5 16 14 16.5 16 13 15 14 17 15 17 14 12.5 11 Anova:Two-Factor With Replication SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5Loc. 6 Loc. 7 Total Desert Eagle Count 10 10 10 10 10 10 10 70 Sum 137141 132.5 134.5 129.5 145 136 955.5 Average 13.7 14.1 13.25 13.45 12.9514.5 13.6 13.65 Variance 1.96 1.88 1.07 0.91 0.14 0.72 0.71 1.20Javalina Count 10 10 10 10 10 10 10 70 Sum 134 134 128.5 132.5 136 130123.5 918.5 Average 13.4 13.4 12.85 13.25 13.6 13 12.35 13.12 Variance2.99 1.16 0.67 1.90 1.60 0.44 0.56 1.37 Midway Count 10 10 10 10 10 1010 70 Sum 149 159 148 152.5 142.5 135.5 139.5 1026 Average 14.9 15.914.8 15.25 14.25 13.55 13.95 14.66 Variance 1.27 0.32 1.51 1.63 1.290.36 1.91 1.64 ANOVA Source of P- Variation SS df MS F value F critVariety 85.22 2 42.61 35.7990 <.0001 3.0437 Location 25.46 6 4.24 3.56580.0023 2.1468 Interaction 40.25 12 3.35 2.8181 0.0014 1.8037 Within224.95 189 1.19 Total 375.88 209 ANOVA shows significant differences (p< .0001) in head length (cm) at harvest maturity stage for variety, andsignificant (p < .01) for location, and the interaction term.Table 10 Presents the Head Width (cm) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert15 14 14 14 14 15 11 Eagle 15 12 15 11 12.5 14 15 13.5 13 15 13 14 15 1412 14 15 14.5 11.5 19 12 14 14 11 14 13.5 16 13 13 15 14 13.5 13.5 15 1315 14 15 13.5 14 15 13 11.5 15 17 12 14 16 13 12 13 15 11.5 14 14 13 1412 17 13 13.5 15 11 Javalina 13.5 15 15 14 13 17 13 15 14 17 14 13 13 1013 12 13 14.5 13 14.5 11 14.5 13 13 12 13 14 13 13 14 14 14 14 13 12 1514 16 15 12 13 14 13 13 15 15.5 14 14.5 13 14 12 17 15 13.5 14 12 14 1216 16.5 13 12.5 13 15 12 15 15 10.5 15 12 Midway 17 15 15 13 14 14 13 1415 15 13 14 17 14 15 14 14 12 16 15 13 15 15 14 14.5 15 17 13 14.5 16 1915 14 15 13 13 15 19 14 12 15 13 17.5 16 17 15 13 14 14 15 14 16 14.5 1314 14 15 16 14 18 13.5 14 15 16 16 14 15 12 14 14 Anova: Two-Factor WithReplication SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7Total Desert Eagle Count 10 10 10 10 10 10 10 70 Sum 135 136 148 130134.5 154 128 965.5 Average 13.5 13.6 14.8 13 13.45 15.4 12.8 13.79Variance 1.78 1.16 2.84 1.33 0.69 2.04 1.51 2.27 Javalina Count 10 10 1010 10 10 10 70 Sum 140 131 151 145.5 129 140.5 123 960 Average 14 13.115.1 14.55 12.9 14.05 12.3 13.71 Variance 0.72 1.21 2.10 1.41 1.04 1.751.34 2.10 Midway Count 10 10 10 10 10 10 10 70 Sum 152 152 157 144 136.5149 136 1026.5 Average 15.2 15.2 15.7 14.4 13.65 14.9 13.6 14.66Variance 1.79 0.62 4.01 2.66 1.56 1.43 0.49 2.21 ANOVA Source of P-Variation SS df MS F value F crit Variety 38.92 2 19.46 12.1993 <.00013.0437 Location 112.23 6 18.71 11.7256 <.0001 2.1468 Interaction 40.1612 3.35 2.0980 0.0188 1.8037 Within 301.50 189 1.60 Total 492.81 209ANOVA shows significant differences (p < .0001) in head width (cm) atharvest maturity stage for variety and location, and significant (p <.05) for the interaction term.Table 11 Presents the Frame Leaf Length (cm) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert26 27 31 20 25 27 26 Eagle 26 26 28 18.5 23 31 31 27 26 29 18 22 30 2618 25 27 17 23 31 29 31 24 26 23 24 31 32 24 27 26 19 22 29 27 29 26 2919 23 30 26 24 26 30 17.5 21 29 30 25.5 27 33 18 21 30 30 30 26 31 23 2331 26 Javalina 33 23 25 21 22 24 28 27 24 30 25 21 22 26 29 22 28 22 2227 21 24 22 21 19.5 22 22 26 27 23 26 26 23 26 24 32 24 29 22 24 20 2626 24 30 18 21 26 25 27 23 35 19 25 27 26 28 22 27 21.5 23 28 27 30 2428 26 26 26 26 Midway 25 24 30 17 23 26 26 35 25 31 18 22 29 26 33 24 2817.5 24 27 28 26 27 33 23.5 23 29 26 28 26 36 23.5 21 28 30 31 24 2816.5 26 22 28 28 24 32 17.5 23 32 29 29 27 34 16 26 25 28 29 25 28 32 2331 28 32 24 28 17.5 21 26 30 Anova: Two-Factor With Replication SUMMARYLoc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Total Desert EagleCount 10 10 10 10 10 10 10 70 Sum 260.5 260 290 193 227 299 283 1812.5Average 26.05 26 29 19.3 22.7 29.9 28.3 25.89 Variance 13.69 0.89 5.334.51 1.57 1.66 5.57 16.68 Javalina Count 10 10 10 10 10 10 10 70 Sum 283231 279 220 229 248 255 1745 Average 28.3 23.1 27.9 22 22.9 24.8 25.524.93 Variance 7.57 0.77 13.43 8.17 2.77 7.07 3.61 10.96 Midway Count 1010 10 10 10 10 10 70 Sum 296 250 308 199 232 275 279 1839 Average 29.625 30.8 19.9 23.2 27.5 27.9 26.27 Variance 9.82 1.56 8.40 25.38 3.078.72 2.32 20.40 ANOVA Source of P- Variation SS df MS F value F critVariety 67.12 2 33.56 5.1872 0.0064 3.0437 Location 1790.51 6 298.4246.1273 <.0001 2.1468 Interaction 301.20 12 25.10 3.8798 <.0001 1.8037Within 1222.73 189 6.47 Total 3381.55 209 ANOVA shows significantdifferences (p < .01) in frame leaf length (cm) at harvest maturitystage for variety; location and the interaction term were alsosignificant at p < .0001.Table 12 Presents the Leaf Width (cm) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert30 28 29 25 25 32 28 Eagle 29 27 26 23 25 33 24 33 28 32 21 29 29 2530.5 27 31 20 31 31 30 32 27 31 29.5 26 32 28 32 28 31 24.5 28 31 28 3827 29 23 27 32 23 25 28 31 19 27 29 28 32 28 33 21.5 29 32 26 33 28 3027 30 32 27 Javalina 29 22 30 23 31 22 29 28 23 33 30 26 23 25 30 21 2521 30 28 26 32 22 28 21.5 28 26 27 30 22 32 34 29 29 28 27 23 33 25.5 2928 29 30 22 31 24 30 22 26 28 21 38 21.5 28 25 24 33 20 28 25 25 28 2833 22 33 30 29 30 24 Midway 35 26 26 16 29 22 28 34 27 24 18 27 27 22 2825 22 19 27 27 24 26 28 24 22 28 26 25 24 28 23 26.5 27 27 21 33 27 2215.5 27 23 28 25 25 27 18 26 28 25 28 28 31 16.5 28 27 21 36 27 23 33.528 31 26 26 26 21 18 25 28 28 Anova: Two-Factor With Replication SUMMARYLoc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Total Desert EagleCount 10 10 10 10 10 10 10 70 Sum 314.5 276 303 233.5 277 313 267 1984Average 31.45 27.6 30.3 23.35 27.7 31.3 26.7 28.34 Variance 11.03 0.273.79 10.50 4.23 1.79 4.68 12.10 Javalina Count 10 10 10 10 10 10 10 70Sum 300 218 311 255.5 285 261 266 1896.5 Average 30 21.8 31.1 25.55 28.526.1 26.6 27.09 Variance 4.44 0.84 12.99 19.30 3.39 8.77 3.60 15.38Midway Count 10 10 10 10 10 10 10 70 Sum 295 267 243 203 272 266 2481794 Average 29.5 26.7 24.3 20.3 27.2 26.6 24.8 25.63 Variance 20.501.34 8.90 32.12 1.29 6.49 7.73 17.53 ANOVA Source of P- Variation SS dfMS F value F crit Variety 258.39 2 129.20 16.1498 <.0001 3.0437 Location1024.95 6 170.82 21.3534 <.0001 2.1468 Interaction 568.34 12 47.365.9203 <.0001 1.8037 Within 1511.98 189 8.00 Total 3363.65 209 ANOVAshows significant differences (p < .0001) in frame leaf width (cm) atharvest maturity stage for variety, location, and the interaction term.Table 13 Presents the Leaf Index (Calculated by Dividing the Leaf Lengthby the Leaf Width)

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert0.87 0.96 1.07 0.80 1.00 0.84 0.93 Eagle 0.90 0.96 1.08 0.80 0.92 0.941.29 0.82 0.93 0.91 0.86 0.76 1.03 1.04 0.59 0.93 0.87 0.85 0.74 1.000.97 0.97 0.89 0.84 0.78 0.92 0.97 1.14 0.75 0.96 0.84 0.78 0.79 0.940.96 0.76 0.96 1.00 0.83 0.85 0.94 1.13 0.96 0.93 0.97 0.92 0.78 1.001.07 0.80 0.96 1.00 0.84 0.72 0.94 1.15 0.91 0.93 1.03 0.85 0.77 0.970.96 Javalina 1.14 1.05 0.83 0.91 0.71 1.09 0.97 0.96 1.04 0.91 0.830.81 0.96 1.04 0.97 1.05 1.12 1.05 0.73 0.96 0.81 0.75 1.00 0.75 0.910.79 0.85 0.96 0.90 1.05 0.81 0.76 0.79 0.90 0.86 1.19 1.04 0.88 0.860.83 0.71 0.90 0.87 1.09 0.97 0.75 0.70 1.18 0.96 0.96 1.10 0.92 0.880.89 1.08 1.08 0.85 1.10 0.96 0.86 0.92 1.00 0.96 0.91 1.09 0.85 0.870.90 0.87 1.08 Midway 0.71 0.92 1.15 1.06 0.79 1.18 0.93 1.03 0.93 1.291.00 0.81 1.07 1.18 1.18 0.96 1.27 0.92 0.89 1.00 1.17 1.00 0.96 1.381.07 0.82 1.12 1.04 1.17 0.93 1.57 0.89 0.78 1.04 1.43 0.94 0.89 1.271.06 0.96 0.96 1.00 1.12 0.96 1.19 0.97 0.88 1.14 1.16 1.04 0.96 1.100.97 0.93 0.93 1.33 0.81 0.93 1.22 0.96 0.82 1.00 1.08 1.23 0.92 1.330.97 0.84 0.93 1.07 Anova: Two-Factor With Replication SUMMARY Loc. 1Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Total Desert Eagle Count 10 1010 10 10 10 10 70 Sum 8.32 9.42 9.60 8.30 8.25 9.57 10.65 64.11 Average0.83 0.94 0.96 0.83 0.82 0.96 1.07 0.92 Variance 0.01 0.00 0.01 0.000.01 0.00 0.01 0.01 Javalina Count 10 10 10 10 10 10 10 70 Sum 9.4910.60 9.01 8.69 8.07 9.60 9.62 65.08 Average 0.95 1.06 0.90 0.87 0.810.96 0.96 0.93 Variance 0.02 0.00 0.01 0.01 0.01 0.02 0.01 0.01 MidwayCount 10 10 10 10 10 10 10 70 Sum 10.22 9.36 12.76 9.87 8.53 10.36 11.3972.50 Average 1.02 0.94 1.28 0.99 0.85 1.04 1.14 1.04 Variance 0.03 0.000.02 0.00 0.00 0.01 0.02 0.03 ANOVA Source of P- Variation SS df MS Fvalue F crit Variety 0.60 2 0.30 31.4353 <.0001 3.0437 Location 1.19 60.20 20.6406 <.0001 2.1468 Interaction 0.84 12 0.07 7.2871 <.0001 1.8037Within 1.81 189 0.01 Total 4.44 209 ANOVA shows significant differences(p < .0001) in frame leaf index at harvest maturity stage for variety,location, and the interaction term.Table 14 Presents the Leaf Area (cm2, Calculated by Multiplying the LeafLength by the Leaf Width)

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert780 756 899 500 625 864 728 Eagle 754 702 728 426 575 1023 744 891 728928 378 638 870 650 549 675 837 340 713 961 870 992 648 806 679 624 992896 768 756 806 466 616 899 756 1102 702 841 437 621 960 598 600 728 930333 567 841 840 816 756 1089 387 609 960 780 990 728 930 621 690 992 702Javalina 957 506 750 483 682 528 812 756 552 990 750 546 506 650 870 462700 462 660 756 546 768 484 588 419 616 572 702 810 506 832 884 667 754672 864 552 957 561 696 560 754 780 528 930 432 630 572 650 756 483 1330409 700 675 624 924 440 756 538 575 784 756 990 528 924 780 754 780 624Midway 875 624 780 272 667 572 728 1190 675 744 324 594 783 572 924 600616 333 648 729 672 676 756 792 517 644 754 650 672 728 828 623 567 756630 1023 648 616 256 702 506 784 700 600 864 315 598 896 725 812 7561054 264 728 675 588 1044 675 644 1072 644 961 728 832 624 588 315 525728 840 Anova: Two-Factor With Replication SUMMARY Loc. 1 Loc. 2 Loc. 3Loc. 4 Loc. 5 Loc. 6 Loc. 7 Total Desert Eagle Count 10 10 10 10 10 1010 70 Sum 8242 7179 8794 4565 6278 9362 7564 51984 Average 824.2 717.9879.4 456.5 627.8 936.2 756.4 742.63 Variance 30376.62 1321.43 9863.1613288.83 2030.84 3950.18 8918.93 32095.87 Javalina Count 10 10 10 10 1010 10 70 Sum 8475 5041 8757 5717.25 6526 6487 6790 47793.25 Average847.5 504.1 875.7 571.725 652.6 648.7 679 682.76 Variance 7550.501385.43 41891.57 29254.26 3863.82 12600.46 6138.00 29436.63 Midway Count10 10 10 10 10 10 10 70 Sum 8748 6686 7526 4290 6317 7360 6917 47844Average 874.8 668.6 752.6 429 631.7 736 691.7 683.49 Variance 29927.073633.60 20835.60 65216.76 3795.34 18087.56 7252.46 35614.93 ANOVA Sourceof Variation SS df MS F P-value F crit Variety 165259.81 2 82629.915.4026 0.0052 3.0437 Location 3022919.57 6 503819.93 32.9415 <.00012.1468 Interaction 789611.91 12 65800.99 4.3023 <.0001 1.8037 Within2890641.78 189 15294.40 Total 6868433.07 209 ANOVA shows significantdifferences (p < .01) for frame leaf area (cm²) at harvest maturitystage in variety, and significance (p < .0001) for location and theinteraction term.Table 15 Presents the Core Length (cm) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert41 40 25 40 40 45 70 Eagle 41 50 25 35 50 50 90 44 40 30 45 45 40 80 3240 30 35 30 45 85 45 40 20 55 50 45 60 40 50 35 35 40 50 70 42 50 20 4545 55 100 27 45 35 35 50 55 70 40 45 15 35 45 45 80 34 50 30 35 45 40 80Javalina 30 90 45 35 40 50 65 43 80 60 30 40 35 65 21 90 35 40 45 45 8018 90 20 25 50 50 65 38 80 50 30 45 55 80 45 90 45 35 35 45 80 28 80 3030 40 65 75 32 90 45 40 45 40 70 44 80 30 55 45 50 65 22 75 35 30 30 4565 Midway 36 160 40 50 45 45 90 65 150 70 40 40 45 100 44 160 20 45 4555 55 36 160 40 45 45 55 75 38 120 40 70 50 45 100 55 98 40 50 45 45 9060 140 40 50 50 50 80 33 150 25 50 40 50 80 45 120 25 60 55 50 90 33 13025 70 50 50 90 Anova: Two-Factor With Replication SUMMARY Loc. 1 Loc. 2Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Total Desert Eagle Count 10 10 10 1010 10 10 70 Sum 386 450 265 395 440 470 785 3191 Average 38.6 45 26.539.5 44 47 78.5 45.59 Variance 32.93 22.22 44.72 46.94 37.78 28.89133.61 268.22 Javalina Count 10 10 10 10 10 10 10 70 Sum 321 845 395 350415 480 710 3516 Average 32.1 84.5 39.5 35 41.5 48 71 50.23 Variance100.77 35.83 135.83 72.22 33.61 67.78 48.89 406.99 Midway Count 10 10 1010 10 10 10 70 Sum 445 1388 365 530 465 490 850 4533 Average 44.5 138.836.5 53 46.5 49 85 64.76 Variance 135.83 449.96 205.83 106.67 22.5015.56 177.78 1278.77 ANOVA Source of P- Variation SS df MS F value Fcrit Variety 14004.18 2 7002.09 75.1698 <.0001 3.0437 Location 82287.986 13714.66 147.2316 <.0001 2.1468 Interaction 34930.82 12 2910.9031.2495 <.0001 1.8037 Within 17605.40 189 93.15 Total 148828.38 209ANOVA shows significant differences (p < .0001) for core length (cm) atharvest maturity stage in variety, location, and the interaction term.Table 16 Presents the Core Diameter (cm) at Harvest Maturity

Trial Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Desert36 30 30 35 40 40 35 Eagle 42 40 35 35 33 40 40 49 35 30 40 35 40 35 3835 30 40 30 35 35 38 30 30 45 30 40 40 32 35 30 4 33 40 35 38 40 30 4 3040 40 30 30 30 35 30 40 35 40 35 30 35 35 40 40 32 40 35 35 30 35 40Javalina 32 40 25 40 35 35 30 30 35 30 30 35 35 30 31 40 25 30 40 35 3521 45 20 30 30 30 35 25 40 35 30 35 30 40 35 40 30 35 35 40 35 30 35 3030 30 35 35 34 40 25 25 35 30 35 35 40 30 35 30 30 30 32 35 35 30 25 3530 Midway 38 40 40 40 35 30 35 40 40 30 30 40 30 30 32 50 30 35 35 40 3033 45 25 40 30 35 30 33 40 30 45 35 35 40 35 40 35 35 30 30 40 33 40 3040 30 30 40 29 45 30 30 30 35 35 35 40 25 45 40 35 40 30 40 25 40 35 3030 Anova: Two-Factor With Replication SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc.4 Loc. 5 Loc. 6 Loc. 7 Total Desert Eagle Count 10 10 10 10 10 10 10 70Sum 375 350 310 308 326 390 375 2434 Average 37.5 35 31 30.8 32.6 3937.5 34.77 Variance 30.94 16.67 4.44 210.62 11.16 4.44 6.94 46.99Javalina Count 10 10 10 10 10 10 10 70 Sum 305 390 285 315 330 335 3352295 Average 30.5 39 28.5 31.5 33 33.5 33.5 32.79 Variance 19.83 10.0022.50 16.94 17.78 11.39 11.39 23.74 Midway Count 10 10 10 10 10 10 10 70Sum 338 420 300 380 340 330 350 2458 Average 33.8 42 30 38 34 33 3535.11 Variance 11.29 12.22 22.22 28.89 15.56 12.22 22.22 29.20 NOVASource of P- Variation SS df MS F value F crit Variety 221.27 2 110.634.4707 0.0127 3.0437 Location 1286.78 6 214.46 8.6664 <.0001 2.1468Interaction 931.33 12 77.61 3.1362 0.0004 1.8037 Within 4677.10 18924.75 Total 7116.48 209 ANOVA shows significant differences (p < .05) incore diameter (mm) at harvest maturity stage for variety, significance(p < .0001) for location, and significance (p < .001) for theinteraction term.Table 17 Presents the Seed Stalk Height (cm)

Desert Eagle Javalina Midway Seed Stalk Height (cm) 100 105 90 90 110 9095 100 95 96 95 85 96 96 80 95 100 81 101 101 82 100 102 75 90 90 78 9595 85 Anova: Single Factor Seed Stalk Height (cm) SUMMARY Groups CountSum Average Variance Desert Eagle 10 958 95.8 14.62 Javalina 10 994 99.432.49 Midway 10 841 84.1 37.88 ANOVA Source of Variation SS df MS FP-value F crit Between Groups 1279.8 2 639.9 22.5877 <.0001 3.3541Within Groups 764.9 27 28.33 Total 2044.7 29 ANOVA shows a significantdifference (p < .0001) in the height (cm) of mature seed stalk.Table 18 Presents the Seed Stalk Spread (cm)

Desert Eagle Javalina Midway Seed Stalk Spread (cm) 45 30 40 40 30 35 4235 40 40 30 42 45 32 45 40 31 40 40 35 35 41 30 38 42 32 36 44 30 40Anova: Single Factor SUMMARY Groups Count Sum Average Variance DesertEagle 10 419 41.9 4.32 Javalina 10 315 31.5 4.06 Midway 10 391 39.110.10 ANOVA Source of Variation SS df MS F P-value F crit Between Groups579.2 2 289.6 47.0186 <.0001 3.3541 Within Groups 166.3 27 6.16 Total745.5 29 ANOVA shows a significant difference (p < .0001) in the spread(cm) of mature seed stalk.

DEPOSIT INFORMATION

A deposit of the lettuce seed of this invention is maintained byShamrock Seed Company Inc., 3 Harris Place, Salinas, Calif. 93901-4593,USA. In addition, a sample of the lettuce seed of this invention hasbeen deposited with the National Collections of Industrial, Food andMarine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, AB243RY, United Kingdom on Sep. 11, 2017.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 C.F.R. 1.801-1.809, Applicants hereby makethe following statements regarding the deposited lettuce cultivar DESERTEAGLE (deposited as NCIMB Accession No. 42806):

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;

4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 C.F.R.1.807; and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of lettuce designated DESERT EAGLE,wherein a representative sample of seed of said lettuce having beendeposited under NCIMB No.
 42806. 2. A lettuce plant, or a part thereofor a plant cell thereof, produced by growing the seed of claim
 1. 3. Thelettuce part of claim 2, wherein the lettuce part is selected from thegroup consisting of: a leaf, a flower, a head, an ovule, pollen and acell.
 4. A tissue culture of regenerable cells produced from the plantor plant part of claim 2, wherein cells of the tissue culture areproduced from a plant part selected from the group consisting ofprotoplasts, embryos, meristematic cells, callus, pollen, ovules,flowers, seeds, leaves, roots, root tips, anthers, stems, petioles,cotyledons and hypocotyls wherein a plant regenerated from the tissueculture has all of the physiological and morphological characteristicsof lettuce DESERT EAGLE listed in Table 1 when grown in the sameenvironmental conditions and wherein a representative sample of seed ofsaid lettuce having been deposited under NCIMB No.
 42806. 5. A lettuceplant regenerated from the tissue culture of claim 4, said plant havingall of the physiological and morphological characteristics of lettuceDESERT EAGLE, wherein a representative sample of seed of said lettucewas deposited under NCIMB No.
 42806. 6. A lettuce head produced from theplant of claim
 2. 7. A method for producing a lettuce head comprising a)growing the lettuce plant of claim 2 to produce a lettuce head, and b)harvesting said lettuce head.
 8. A lettuce head produced by the methodof claim
 7. 9. A method for producing a lettuce seed comprising crossinga first parent lettuce plant with a second parent lettuce plant andharvesting the resultant lettuce seed, wherein said first parent lettuceplant and/or second parent lettuce plant is the lettuce plant of claim2.
 10. The lettuce seed produced by the method of claim
 9. 11. A methodfor producing a lettuce seed comprising self-pollinating the lettuceplant of claim 2 and harvesting the resultant lettuce seed.
 12. Alettuce seed produced by the method of claim
 11. 13. A method ofproducing a lettuce plant derived from the lettuce DESERT EAGLE, themethod comprising the steps of: (a) crossing the plant of claim 2 with asecond lettuce plant to produce a progeny plant; (b) crossing theprogeny plant of step (a) with itself or a second lettuce plant toproduce a seed; (c) growing a progeny plant of a subsequent generationfrom the seed produced in step (b); (d) crossing the progeny plant of asubsequent generation of step (c) with itself or a second lettuce plantto produce a lettuce plant derived from the lettuce DESERT EAGLE. 14.The method of claim 13 further comprising the step of: (d) repeatingstep b) and/or c) for at least 1 more generation to produce a lettuceplant derived from the lettuce DESERT EAGLE.
 15. The plant of claim 2comprising at least one single locus conversion and otherwiseessentially all of the physiological and morphological characteristicsof lettuce Desert Eagle listed in Table 1 when grown under the sameenvironmental conditions.
 16. The plant of claim 15 wherein the at leastone single locus conversion confers said plant with a trait selectedfrom the group consisting of male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, heat tolerance, improved shelf life, delayed shelf life, andimproved nutritional quality.
 17. The plant of claim 16 wherein the atleast one single locus conversion is an artificially mutated gene ornucleotide sequence.
 18. A lettuce plant, having all of thephysiological and morphological characteristics of lettuce DESERT EAGLElisted in Table 1 when grown in the same environmental conditions, or apart or a plant cell thereof.
 19. A lettuce plant, or a part thereof,having all of the physiological and morphological characteristics oflettuce DESERT EAGLE, wherein a representative sample of seed of saidlettuce having been deposited under NCIMB No.
 42806. 20. A method ofintroducing a desired trait into lettuce DESERT EAGLE comprising: (a)crossing a lettuce DESERT EAGLE plant grown from lettuce DESERT EAGLEseed, wherein a representative sample of seed has been deposited underNCIMB No. 42806, with another lettuce plant that comprises a desiredtrait to produce F1 progeny plants, wherein the desired trait isselected from the group consisting of insect resistance, herbicideresistance, disease resistance, water stress tolerance, heat tolerance,improved shelf life, delayed shelf life, and improved nutritionalquality; (b) selecting one or more progeny plants that have the desiredtrait to produce selected progeny plants; (c) crossing the selectedprogeny plants with the lettuce DESERT EAGLE plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait and the physiological and morphological characteristics oflettuce DESERT EAGLE listed in Table 1 when grown in the sameenvironmental conditions to produce selected backcross progeny plants;and (e) repeating steps (c) and (d) three or more times in succession toproduce selected fourth or higher backcross progeny plants that comprisethe desired trait and the physiological and morphologicalcharacteristics of lettuce DESERT EAGLE listed in Table 1 when grown inthe same environmental conditions.